Pharmaceutical composition for treatment of parkinson's disease, containing sitagliptin as active ingredient

Abstract

The present invention relates to a pharmaceutical composition for treatment of Parkinson's disease, containing sitagliptin as an active ingredient. It has been identified that, among patients with Parkinson's disease with comorbid diabetes, a sitagliptin-administered group exhibited less dopaminergic neuron loss at the time of diagnosis in comparison to a non-administered group, and in comparison even to a group without diabetes, the loss of dopaminergic neurons was milder. It has been also identified that oral administration of the sitagliptin in an animal model of Parkinson's disease improves intestinal inflammation and microbial groups, alleviates deposition of α-synuclein in the intestine, and alleviates deposition of α-synuclein in brain tissue, and thus the sitagliptin is provided as a novel treatment for Parkinson's disease on the basis of the gut-brain axis.

Description

Pharmaceutical composition for the treatment of Parkinson's disease containing sitagliptin as an active ingredient

[0001] The present invention relates to a pharmaceutical composition for treating Parkinson's disease, comprising sitagliptin as an active ingredient.

[0002] Parkinson's disease is the second most common neurodegenerative disease after Alzheimer's disease, with a prevalence of 1% in those over 60 years of age, and its incidence is known to increase with age. The prevalence and incidence of Parkinson's disease in Korea are not yet precisely known, but as of 2020, it was estimated to be 111,311. Parkinson's disease is characterized by four major symptoms: bradykinesia, rigidity, tremor, and postural imbalance, due to abnormal intracellular aggregation / deposition of alpha-synuclein proteins, which causes nerve cell death and neurological dysfunction. Aggregated alpha-synuclein spreads to microglia, inducing / promoting a neuroinflammatory response and accelerating cell death.

[0003] A total of 38 products have been approved for the treatment of Parkinson's disease in Korea, including 4 products containing levodopa / benserazide, 5 products containing a combination of levodopa / carbidopa, 4 products containing amantadine, 1 product containing selegiline hydrochloride, 2 products containing entacapone, and 22 products containing levodopa / carbidopa / entacapone. The current treatment for Parkinson's disease is limited to symptom control through dopamine supplements, and dopamine treatment has limitations as a treatment because the efficacy decreases as the disease progresses and side effects such as dyskinesia and loss of efficacy appear. No successful disease-modifying agent has been reported worldwide to slow the progression of Parkinson's disease. Given the progressive nature of this degenerative brain disease, controlling its progression is expected to be a fundamental treatment for the disease. In particular, research is ongoing into fundamental therapeutic approaches that suppress aggregation / deposition and cell-to-cell transmission of alpha-synuclein, a protein recognized as a cause of Parkinson's disease.

[0004] Meanwhile, the 'gut-brain axis' has been reported to be an important pathophysiology in Parkinson's disease. The hypothesis that alpha-synuclein protein aggregates / deposits in the gut plexus and spreads intercellularly through the vagus nerve to the medulla oblongata, pons, midbrain, and cerebrum, causing neuroinflammation and neurodegeneration has been confirmed through various studies. When the autopsy findings of Parkinson's disease patients were comprehensively reviewed, it was confirmed that alpha-synuclein aggregates / deposit in the medulla oblongata, pons, midbrain, and cerebrum in that order depending on the severity of the disease. Based on this, the 'gut-brain axis', in which alpha-synuclein protein aggregates / deposit in the gut plexus and spreads intercellularly through the vagus nerve to the medulla oblongata, pons, midbrain, and cerebrum, causing neuroinflammation and neurodegeneration, has been noted as a cause of Parkinson's disease.

[0005] The purpose of the present invention is to provide a pharmaceutical composition for preventing or treating Parkinson's disease, which comprises sitagliptin or a pharmaceutically acceptable salt thereof as an active ingredient, and to provide a new means of treating Parkinson's disease based on the gut-brain axis.

[0006] The present invention provides a pharmaceutical composition for preventing or treating Parkinson's disease, comprising sitagliptin or a pharmaceutically acceptable salt thereof as an active ingredient.

[0007] In addition, the present invention provides a reagent composition for inhibiting α-synuclein deposition in the intestine, vagus nerve, medulla oblongata or midbrain of an animal model of Parkinson's disease other than a human, comprising sitagliptin as an active ingredient.

[0008] The present invention also provides a method for inhibiting α-synuclein deposition in the intestine, vagus nerve, medulla oblongata, or midbrain, comprising administering sitagliptin to an animal model of Parkinson's disease other than a human.

[0009] According to the present invention, in patients with Parkinson's disease accompanied by diabetes, the group taking sitagliptin showed less loss of dopaminergic neurons at the time of diagnosis compared to the group not taking sitagliptin, and the loss of dopaminergic neurons was even milder compared to the group without diabetes, and in an animal model of Parkinson's disease, oral administration of sitagliptin not only improved intestinal inflammation and microbiota, but also improved intestinal synuclein (α-synuclein) deposition and synuclein (α-synuclein) deposition in brain tissue, so sitagliptin can be provided as a new treatment for Parkinson's disease based on the gut-brain axis.

[0010] Figure 1 shows a flow chart of a database analysis of Parkinson's disease patients according to the present invention.

[0011] Figure 2 is a schematic diagram of an experiment to evaluate the effect of sitagliptin in an animal model of Parkinson's disease.

[0012] Figure 3 shows the results of analyzing the availability of dopamine transporter (DAT) in the striatal subregion in a database of Parkinson's disease patients.

[0013] Figure 4 shows the results of evaluating longitudinal changes in levodopa-equivalent doses (LED) in a database of patients with Parkinson's disease.

[0014] Figure 5 shows the results of analyzing Kaplan-Meier survival curves for levodopa-induced dyskinesia (LID) and wearing-off (WO) in a database of patients with Parkinson's disease.

[0015] Figure 6a shows the results of analyzing body weight changes from week 1 to week 12 in an animal model of Parkinson's disease.

[0016] Figure 6b shows the results of measuring the intestinal transit distance and colon length using Evans blue at week 12 of the experiment in an animal model of Parkinson's disease.

[0017] Figure 6c shows the results of analyzing representative Western blot bands and intensities of inflammatory markers Iba-1, TLR2, TLR4, CD3, and IL-1β in an animal model of Parkinson's disease.

[0018] Figure 6d shows the results of immunohistochemical analysis of the expression levels of Iba-1, TLR2, CD3, and IL-1β in an animal model of Parkinson's disease.

[0019] Figure 6e shows the results of analyzing serum cytokine (TNF-α, IFN-γ, IL-1β, and IL-6) levels in an animal model of Parkinson's disease.

[0020] Figure 6f shows the results of Western blot and intensity analysis of the expression levels of tight junction markers (Claudin1, Claudin5, and Occludin) in an animal model of Parkinson's disease.

[0021] Figure 7a shows the results of an immunohistochemical analysis of the effect of sitagliptin on α-synuclein aggregation in the intestinal mucosa and muscle layer in an animal model of Parkinson's disease.

[0022] Figure 7b shows the results of analyzing the effect of sitagliptin on α-synuclein aggregation in the intestinal mucosa and muscle layer in an animal model of Parkinson's disease using 3,3-diaminobenzidine (DAB) intensity.

[0023] Figure 7c shows the results of immunofluorescence analysis of α-synuclein and EEC (enteroendocrine cell) markers (PYY, GLP-1, Ngn1, and TH) to determine the effect of sitagliptin on α-synuclein aggregation in the intestinal mucosa and muscle layer in an animal model of Parkinson's disease.

[0024] Figure 8a shows that after STC-1 cells were treated with 25, 50, 100, and 250 nM rotenone for 48 hours, the protein expression of α-syn and cleaved PARP was analyzed and quantified by Western blotting.

[0025] Figure 8b shows the results of Western blotting analysis of α-syn protein expression after treatment with 100 nM rotenone in Caco-2 and STC-1 cells.

[0026] Figure 8c shows the results of immunofluorescence staining analysis of α-syn protein expression after treatment with 100 nM rotenone in Caco-2 and STC-1 cells.

[0027] Figure 8d is a graph showing the results of quantifying the levels of intracellular α-syn and extracellular α-syn present in the culture medium of Ctrl (control group), Rot (rotenone treatment group), and Rot+Sita (rotenone and sitagliptin simultaneous treatment group).

[0028] Figure 8e shows the results of co-treatment of Raw264.7 cells with 1 μg / mL of Pam3CSK4 and 100 μM sitagliptin for 24 hours. The expression of iNOS and COX2 was analyzed by Western blotting and presented quantitatively in a graph.

[0029] Figure 8f shows the results of co-treatment of enteric glial cells (EGCs) with 50 μM rotenone and 100 μM sitagliptin for 24 hours. The expression of iNOS and COX2 was analyzed by Western blotting and quantitatively represented in a graph.

[0030] Figure 8g shows that STC-1 cells were simultaneously treated with 100 nM rotenone and 25 μM sitagliptin for 24 hours, and the expression of iNOS and COX2 was analyzed by Western blotting and presented as a quantitative graph.

[0031] Figure 9a presents p-α-syn immunostaining images and quantitative analysis results from vagus nerve sections 3 months after rotenone administration (right panel, n=6). Magnified images of p-α-syn-stained vagus nerve cell bodies (arrows) and Schwann cells (arrowheads) are shown.

[0032] Figure 9b is an immunofluorescence image simultaneously staining p-α-syn, neurofilament (NF), and S100β.

[0033] Figure 9c presents p-α-syn staining images of the left and right dorsal vagal nuclei (DMV) at 3 months of rotenone administration and shows the results of quantitative analysis.

[0034] Figure 9d is an immunofluorescence image showing the colocalization of p-α-syn and choline acetyltransferase (ChAT) in the dorsal vagal nucleus (DMV). Double staining with p-α-syn (green) and ChAT (red) is performed, and the yellow arrow indicates the DMVM region containing ChAT-positive cells.

[0035] Figure 9e presents images of p-α-syn staining in the substantia nigra pars compacta (SNpc) 6 months after rotenone administration and shows the results of quantitative analysis.

[0036] Figure 9f presents an immunofluorescence double staining image of p-α-syn (green) and tyrosine hydroxylase (TH, red) in SNc.

[0037] Figure 10a shows the protein expression of ChAT in the medulla oblongata 6 months after rotenone administration, evaluated through Western blotting analysis, and the quantitative results are expressed as the mean ± standard deviation.

[0038] Figure 10b shows the protein expression of TH in the midbrain at 6 months after rotenone administration, evaluated by Western blotting analysis, and the quantitative results are expressed as the mean ± standard deviation.

[0039] Figure 10c shows a graph showing TH immunostaining images and quantitative analysis 6 months after rotenone administration.

[0040] Figure 10d presents immunostaining images of Iba-1, a microglial marker, in the substantia nigra pars compacta (SNpc) at 6 months of rotenone administration (first to third panels). These images were converted to binary images (fourth panel) for quantitative analysis. The number of Iba-1-positive microglia (left graph) and average cell size (right graph) were measured and presented.

[0041] Figure 10e shows that motor function was evaluated by performing the Rotarod test at a constant speed (30 rpm) and acceleration conditions (5-40 rpm) 6 months after rotenone administration.

[0042] Figure 11a shows the results of analyzing the effect of sitagliptin on the intestinal microbiota in an animal model of Parkinson's disease using the alpha diversity of intestinal microbiota through ASV and Shannon index.

[0043] Figure 11b shows the results of a PCoA plot of beta diversity based on unweighted and weighted UniFrac PERMANOVA analyses of various groups to analyze the effects of sitagliptin on the gut microbiota in an animal model of Parkinson's disease.

[0044] Figure 11c shows the results of analyzing the effects of sitagliptin on the gut microbiota in an animal model of Parkinson's disease by the ratio of Firmicutes to Bacteroidetes between different groups at the taxonomic level of phylum.

[0045] Figure 11d shows the results of analyzing the effect of sitagliptin on the gut microbiota in an animal model of Parkinson's disease by the relative abundance of gut microbes at the genus level.

[0046] Figure 11e shows the results of analyzing the relative abundance of five bacterial genera (Clostridium, Prevotella, Alistipes, Mediterraneibacter, and Ruminiclostridium) to determine the effect of sitagliptin on the gut microbiota in an animal model of Parkinson's disease.

[0047] The terms used in this specification have been selected from widely used, current terms, taking into account the functions of the present invention. However, these terms may vary depending on the intentions of those skilled in the art, precedents, the emergence of new technologies, etc. Furthermore, in certain cases, terms may be arbitrarily selected by the applicant, and in such cases, their meanings will be described in detail in the relevant description of the invention. Therefore, the terms used in this invention should not be defined simply as names, but rather based on their inherent meanings and the overall content of the present invention.

[0048] Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Terms defined in commonly used dictionaries should be interpreted as having a meaning consistent with their meaning in the context of the relevant technology, and shall not be interpreted in an idealized or overly formal sense unless explicitly defined herein.

[0049]

[0050] Hereinafter, the present invention will be described in more detail.

[0051] The present invention provides a pharmaceutical composition for preventing or treating Parkinson's disease, comprising sitagliptin or a pharmaceutically acceptable salt thereof as an active ingredient.

[0052] The above sitagliptin is a compound represented by the following chemical formula 1, and its IUPAC name is (R)-4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl]-1-(2,4,5-trifluorophenyl)butan-2-amine, and its CAS number is 486460-32-6. The above sitagliptin is an oral hypoglycemic agent that extends the half-life of GLP-1 and GIP by inhibiting the enzymatic degradation and inactivation of incretin.

[0053] [Chemical Formula 1]

[0054]

[0055] Preferably, the Parkinson's disease may be Parkinson's disease accompanied by type 2 diabetes, but is not limited thereto.

[0056] Preferably, the sitagliptin can suppress intestinal inflammatory response, suppress α-synuclein deposition in the intestine, vagus nerve, medulla oblongata or midbrain, suppress neuronal loss in the medulla oblongata and midbrain, and restore motor ability, but is not limited thereto.

[0057] Preferably, the pharmaceutical composition has an effect of improving the intestinal microflora of the patient with Parkinson's disease, and more preferably, the effect of improving the intestinal microflora may be, but is not limited to, a decrease in the abundance of intestinal microorganisms belonging to the genera Prevotella, Alistipes, and Mediterraneibacter.

[0058] The above pharmaceutically acceptable salt means an acid addition salt formed by a pharmaceutically acceptable free acid, and the pharmaceutically acceptable salt means a salt commonly used in the pharmaceutical industry, for example, an inorganic ion salt manufactured with calcium, potassium, sodium or magnesium, etc.; an inorganic acid salt manufactured with hydrochloric acid, nitric acid, phosphoric acid, hydrobromic acid, iodic acid, perchloric acid or sulfuric acid, etc.; an organic acid salt manufactured with acetic acid, trifluoroacetic acid, citric acid, maleic acid, succinic acid, oxalic acid, benzoic acid, tartaric acid, fumaric acid, mandelic acid, propionic acid, lactic acid, glycolic acid, gluconic acid, galacturonic acid, glutamic acid, glutaric acid, glucuronic acid, aspartic acid, ascorbic acid, carbonic acid or vanillic acid, etc.; There are sulfonic acid salts manufactured from methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, etc.; amino acid salts manufactured from glycine, arginine, lysine, etc.; or amine salts manufactured from trimethylamine, triethylamine, ammonia, pyridine, picoline, etc.; but the types of salts meant in the present invention are not limited by these listed salts.

[0059] The pharmaceutical composition of the present invention can be manufactured in a unit dose form or can be manufactured by placing it in a multi-dose container by formulating it using a pharmaceutically acceptable carrier according to a method that can be easily performed by a person having ordinary skill in the art to which the present invention pertains.

[0060] The pharmaceutically acceptable carriers mentioned above are those commonly used in formulations, and include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil. In addition to the above components, the pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like.

[0061] In the present invention, the content of the additive included in the pharmaceutical composition is not particularly limited and can be appropriately adjusted within the content range used in conventional formulations.

[0062] The above pharmaceutical composition may be formulated in the form of one or more external preparations selected from the group consisting of injectable formulations such as aqueous solutions, suspensions, emulsions, pills, capsules, granules, tablets, creams, gels, patches, sprays, ointments, ointments, lotions, liniments, pastes, and cataplasmas.

[0063] The pharmaceutical composition of the present invention may further comprise pharmaceutically acceptable carriers and diluents for formulation. The pharmaceutically acceptable carriers and diluents include, but are not limited to, excipients such as starches, sugars, and mannitol; fillers and extenders such as calcium phosphate; cellulose derivatives such as carboxymethylcellulose and hydroxypropylcellulose; binders such as gelatin, alginates, and polyvinyl pyrrolidone; lubricants such as talc, calcium stearate, hydrogenated castor oil, and polyethylene glycol; disintegrants such as povidone and crospovidone; and surfactants such as polysorbates, cetyl alcohol, and glycerol. The pharmaceutically acceptable carriers and diluents may be biologically and physiologically compatible with the subject. Examples of diluents include, but are not limited to, saline, aqueous buffers, solvents, and / or dispersion media.

[0064] The pharmaceutical composition of the present invention is for oral administration and may be formulated as a tablet, troches, lozenge, aqueous suspension, oily suspension, prepared powder, granules, emulsion, hard capsule, soft capsule, syrup or elixir.

[0065] The dosage of the pharmaceutical composition of the present invention may vary depending on the patient's condition and weight, age, sex, health status, dietary constitution, nature of the formulation, severity of the disease, administration time of the composition, administration method, administration period or interval, excretion rate, and drug form, and may be appropriately selected by a person skilled in the art. For example, the dosage may range from about 0.1 to 10,000 mg / kg, but is not limited thereto, and may be administered once or several times a day in divided doses.

[0066] The pharmaceutically effective amount and effective dosage of the pharmaceutical composition of the present invention may vary depending on the method of formulation, administration method, administration time, and / or administration route of the pharmaceutical composition. Those skilled in the art can easily determine and prescribe an effective dosage for the desired treatment. The pharmaceutical composition of the present invention may be administered once a day or divided into several doses.

[0067]

[0068] In addition, the present invention provides a reagent composition for inhibiting α-synuclein deposition in the intestine, vagus nerve, medulla oblongata or midbrain of an animal model of Parkinson's disease other than a human, comprising sitagliptin as an active ingredient.

[0069] The present invention also provides a method for inhibiting α-synuclein deposition in the intestine, vagus nerve, medulla oblongata, or midbrain, comprising administering sitagliptin to an animal model of Parkinson's disease other than a human.

[0070] Below, the present invention is described in detail using examples that do not limit the scope of the invention. The following examples are intended to concretize the invention and do not limit or restrict the scope of the invention. Therefore, anything that a specialist in the technical field can easily infer from the detailed description and examples of the invention is interpreted as falling within the scope of the invention.

[0071]

[0072] Materials and Experimental Methods

[0073] [Experimental Example 1] Clinical Data

[0074] Target audience

[0075] A database of 719 de novo Parkinson's disease (PD) patients who visited the Movement Disorders outpatient clinic at Severance Hospital from April 2009 to December 2016 was reviewed, provided by the Yonsei Parkinson Center. PD was diagnosed according to the clinical diagnostic criteria of the UK PD Society Brain Bank. 18F-FP-CIT PET (N-(3-[18F]fluoropropyl)-2β-carbon ethoxy-3β-(4-iodophenyl) nortropane) scans showed decreased dopamine transporter (DAT) availability in the posterior putamen in all subjects. Patients with drug-induced Parkinson's disease, atypical Parkinson's disease, bedridden patients due to other disorders, and drug-naïve Parkinson's disease were excluded from the analysis subjects (Fig. 1). Parkinson's disease motor symptoms were assessed using the Unified PD Rating Scale Part III (UPDRS-III) at the initial visit in a dopamine naïve state, and olfactory function was measured using the cross-cultural smell identification test (CCSIT). Depression was assessed using the Beck Depression Inventory (BDI), and general cognition was assessed using the Korean version of the Mini-Mental State Examination (K-MMSE). PD patients were classified into tremor-dominant or postural instability / gait disturbance parkinsonism based on clinical phenotype. All patients were examined for medical history other than type 2 diabetes mellitus (DM) and vascular risk factors such as hypertension, dyslipidemia, heart disease, and ischemic stroke. New-onset diabetes was defined as a record of a physician-diagnosed diabetes, a record of initiating diabetes treatment, or a record of fasting blood glucose >125 mg / dL or HbA1c ≥6.5% in those without a history of diabetes, based on the electronic medical records of Severance Hospital. Parkinson's disease (PD) was classified into the following three groups based on the presence of type 2 diabetes mellitus (DM) and treatment with dipeptidyl peptidase-4 inhibitors (DPP-4i).

[0076] 1) PD patients without diabetes (PD-DM) - group, n = 558)

[0077] 2) Patients with PD diabetes not treated with DPP-4i (PD-DM) + / DPP-4i - group, n = 85)

[0078] 3) Patients with PD diabetes receiving DPP-4i treatment (PD-DM) + / DPP-4i + group, n = 54)

[0079] We investigated whether patients were prescribed oral hypoglycemic agents and insulin other than DPP-4i. In addition, we investigated the type of antidiabetic medication and DPP-4i they were taking, as follows: sitagliptin (n = 30), linagliptin (n = 10), vildagliptin (n = 6), gemigliptin (n = 3), alogliptin (n = 2), saxagliptin (n = 2), and teneligliptin (n = 1).

[0080] There were no PD diabetic patients receiving treatment with GLP-1 agonists. This study was approved by the Yonsei University Severance Hospital Institutional Review Board (IRB No. 4-2014-0637), and the need for informed consent was waived due to its retrospective nature.

[0081]

[0082] 18 F-FP-CIT PET scan acquisition and quantitative analysis

[0083] 18F-FP-CIT PET scans were acquired using a GE PET-CT DSTe scanner (GE Discovery STE, GE Healthcare, Milwaukee, WI, USA), which produced images with a three-dimensional resolution of 2.3 mm at half maximum width. Subjects fasted for at least 6 hours, and then 18 F-FP-CIT 5 mCi (185 MBq) was injected intravenously. PET images were acquired 90 minutes after injection in three-dimensional mode at 12 kVp and 380 mA for 20 minutes. Image processing was performed using SPM8 (Wellcome Department of Imaging Neuroscience, Institute of Neurology, UCL, London, UK) with Matlab 2013a for Windows (Math Works, Natick, MA, USA). Quantitative analysis was performed based on volumes of interest (VOI) defined based on a template in standard space. All reconstructed PET images were generated from standard 18F-FP-CIT PET. 18 The F-FP-CIT PET template and the weighted MRI scans of 13 normal controls were spatially normalized to the Montreal Neurology Institute (MNI) template space. Co-registered spatially normalized single T1-weighted MR and MRIcro version 1.37 (Chris Rorden, Columbia, SC, USA) 18Twelve volumes of interest (VOIs) in the bilateral striatal subregions and one occipital VOI were drawn on the F-FP-CIT PET template image. The striatum was divided into dorsal and ventral parts along the anteroposterior commissure in the transverse plane. The ventral portion consisted of two subregions: the ventral putamen and the ventral striatum. The dorsal portion was divided into the anterior caudate, posterior caudate, anterior putamen, and posterior putamen subregions along the coronal anterior commissure plane. The VOIs were adjusted using the editing software ANIQUE. DAT availability was calculated by the irreplaceable binding potential defined as: [mean normalized uptake value of striatal subregions VOI - mean normalized uptake value of occipital VOI] / [mean normalized uptake value of occipital VOI].

[0084]

[0085] FLAIR sequence image acquisition and white matter hyperintensity grading

[0086] Of the 697 enrolled patients, 527 (75.6%) underwent brain MRI scans at Severance Hospital using a 3.0T scanner (Achieva, Philips Medical System, Best, Netherlands) including fluid-attenuated inversion recovery (FLAIR) sequence images at the initial evaluation. FLAIR sequence images were acquired using the following parameters: matrix, 512 × 512; slice number, 22; pixel spacing, 0.449 × 0.449 mm2; slice thickness, 5 mm; gap, 2 mm; field of view, 230 mm; repetition time, 11,000 ms; echo time, 125 ms; inversion time, 2,800 ms; flip angle, 90°. The remaining 170 patients (24.4%) underwent brain MRI examinations including FLAIR images. A visual grading scale for white matter hyperintensities (WMH) was assessed by two neurologists using the Scheltens scale, which semiquantitatively assesses periventricular and lobar white matter (frontal, parietal, temporal, and occipital) hyperintensities, basal ganglia, and infratentorial signal hyperintensities. The reliability of the total WMH assessment was confirmed to be high, with intraclass correlation coefficients of 0.984 and 0.966, respectively. The final consensus rating between the two raters was used in the analysis.

[0087]

[0088] Longitudinal assessment of changes in levodopa equivalent daily dose over time.

[0089] Among a total of 697 PD patients, 605 PD patients (PD-DM) who received dopaminergic drug treatment for at least 2 years - , n = 483; PD-DM + / DPP-4i - , n = 76; PD-DM + / DPP-4i + , n = 46) were included in the longitudinal evaluation of levodopa-equivalent doses (LED) (Fig. 1). Patients received outpatient treatment every 3 to 6 months, and the median number of visits was 20 (range: 6 to 56). Dopaminergic drugs were prescribed by specialists according to patient response for effective symptom control. The dose of dopaminergic drugs was checked at each visit, and the levodopa-equivalent dose (LED) was calculated using the following mathematical formula.

[0090] [Mathematical Formula 1]

[0091] LED = Levodopa × 1 + Controlled-release levodopa × 0.75 + Ropinirole × 20 + Pramipexole × 100 + Levodopa × 0.33 (Entacapone + Selegiline × 10 + Rasagiline × 100)

[0092]

[0093] A linear mixed model was used to compare longitudinal changes in LED between groups. The model included nine fixed effects: eight subject effects, including age at symptom onset, sex, disease duration, hypertension, dyslipidemia, BMI, total WMH, and baseline DAT availability in the posterior putamen, by PD group, and a within-subject effect time. Because most increases in LED occurred within the first 6 months and dopaminergic medication doses were adjusted at 3- to 6-month intervals thereafter, time was treated as a categorical variable in 6-month intervals up to a maximum of 60 months (>40% of patients were followed). The effect of PD group on longitudinal changes in LED was tested using a group × time interaction term.

[0094]

[0095] Assessment of the incidence of levodopa-induced dyskinesia and weakness

[0096] The subjects underwent outpatient care every 3 to 6 months, and the presence of levodopa-induced dyskinesia (LID) and fatigue were assessed by two movement disorder specialists based on medical histories provided by patients and their caregivers and direct neurological examination at each visit. The date of LID or fatigue was considered the date when the PD patient or their caregiver reported the onset of LID or fatigue, or when LID or fatigue was first observed in the clinic. The time from treatment initiation to LID onset and fatigue was estimated using Kaplan-Meier estimates in 606 PD patients with a follow-up period of 2 years or longer (Fig. 5). The log-rank test was used to compare Kaplan-Meier plots between groups. To assess the effect of DPP-4i on the development of LID and fatigue, Cox regression models were used to estimate hazard ratios (HRs) and 95% confidence intervals (CIs), adjusting for age at symptom onset, sex, disease duration, hypertension, dyslipidemia, total WMH, and baseline DAT availability in the posterior putamen. For patients with dyskinesia, levodopa doses were administered per body weight at the onset of LID or fatigue or at the last outpatient clinic visit for patients without dyskinesia.

[0097]

[0098] Statistical analysis

[0099] To compare baseline demographic characteristics between PD groups, one-way analysis of variance with post hoc Bonferroni correction was used for continuous variables, and Pearson's χ2 test or Fisher's exact test was used for categorical variables. Independent-samples t-tests were used to compare the duration of DM between the PD groups. Bonferroni correction was used for multiple comparisons to compare DAT availability in each striatal subregion between the PD groups. Multivariate linear regression analysis was used to determine the independent effect of DPP-4i after adjusting for age at symptom onset, sex, disease duration, hypertension, dyslipidemia, BMI, and total WMH. Linear mixed models were used to compare longitudinal LED changes between the three PD groups. The effects of DPP-4i on the development of LID and fatigue were assessed using the log-rank test and Cox regression model, as described above. Statistical analyses were performed using SPSS (version 25.0, IBM Corporation, Armonk, NY, USA) and R (v3.6, https: / / www.r-project.org / ). Results with a two-sided P < 0.05 were considered statistically significant.

[0100]

[0101] [Experimental Example 2] Experimental Data

[0102] laboratory animals

[0103] C57BL / 6J mice (male, 8 weeks old) were purchased from Orient Bio. All mice were maintained in a specific pathogen-free facility at the Yonsei Laboratory Animal Research Center. Animals were provided free access to food and water and were maintained under climate- and lighting-controlled conditions (24±0.5°C, 12-h / 12-h light / dark cycle). This experiment was conducted under the approval of the Institutional Animal Care and Use Committee of Yonsei University Health System (IACUC approval number YUHS-IACUC-2022-0251).

[0104]

[0105] Rotenone-induced mouse model and treatment

[0106] Mice were randomly divided into three groups: (a) control (Ctrl) group, (b) rotenone (Rot) group, and (c) rotenone + sitagliptin (Rot + Sita) group. Rotenone was treated to prepare an animal model of Parkinson's disease. Rotenone was administered to the mice by oral gavage at a dose of 10 mg / kg / day in a vehicle solution containing 0.5% carboxymethylcellulose (CMC-Na; S6703, Selleckchem) and 1.25% chloroform daily. The Rot + Sita group was co-administered with sitagliptin (S5079, Selleckchem) at a dose of 30 mg / kg / day. The control group was administered the vehicle solution. Mice were sacrificed 4, 12, and 24 weeks after the start of rotenone administration, and brain, intestinal tissue, and blood were collected for analysis (Fig. 2).

[0107]

[0108] Intestinal transit and colon length

[0109] Mice were orally administered 0.3 mL of 2.5% Evans blue (Sigma-Aldrich) suspended in 1.5% CMC-Na2 20 minutes prior to sacrifice. Intestinal transit after euthanasia was measured as the distance of the dye from the pylorus to its furthest point of travel. Total colonic length was measured from the end of the cecum to the anus.

[0110]

[0111] Measurement of proinflammatory cytokines

[0112] Blood was collected from the abdominal aorta in BD Microtainer SST™ tubes (BD Diagnostics) and centrifuged at 3000 rpm for 10 min at 4°C to collect serum. The mouse proinflammatory cytokine multiplex ELISA kit (IL-1β, IFN-γ, TNF-α, IL-6) was purchased from Arigobio (ARG82842), and the expression of all serum cytokines was measured according to the manufacturer's instructions.

[0113]

[0114] Immunohistochemistry

[0115] Mice were anesthetized with isoflurane and transcardially perfused with 4% paraformaldehyde (PFA; BYLABS). Tissues were embedded in paraffin and sectioned at 5 μm thickness. After deparaffinization and rehydration, antigen retrieval was performed using IHC Tek™ Epitope Retrieval Solution (IHC WORLD) and boiling the slides in plastic Coplin jars for 20 minutes (95°C). After cooling to room temperature for 10 minutes, endogenous peroxidase and mouse IgG were blocked with 3% H2O2 in methanol and Mouse-On-Mouse blocking reagent (Vector Laboratories), respectively. The slides were incubated with 1% bovine serum albumin in PBST for 1 hour and then reacted with primary antibodies overnight at 4°C. The primary antibodies used were as follows: anti-α-synuclein (ab212184, Abcam), anti-α-synuclein (610787, BD Biosciences), anti-phosphor-α-synuclein (ab51253, Abcam), anti-TH (T2928, Sigma-Aldrich), anti-TLR2 (#MA5-32787, Invitrogen), anti-TLR4 (#MA5-16216, Invitrogen), anti-Iba-1 (ab178847, abcam), anti-CD3 (ab16669, abcam), anti-IL-1β (sc-52012, Santa-cruz), anti-PYY (ab22663, abcam), anti-neurogenin 1 (MBS854065, Mybiosource), anti-GLP-1 (ab11125), anti-ocludin (#91131, Cell signaling), anti-claudin 1 (#37-4900, Invitrogen), anti-claudin 5 (#35-2500, Invitrogen) and anti-ChAT (MA5-31383, Invitrogen).Slides were washed and incubated with peroxidase-conjugated secondary antibodies for 1 hour at room temperature. Sections were washed three times with PBST buffer and reacted with 3,3-diaminobenzidine (DAB) using a DAB substrate kit (Vector Laboratories). Slides were lightly counterstained with Mayer's hematoxylin (DAKO). Machine learning-based software (ZEISS Microscopy, ZEN Intellesis) was used for image segmentation from the colon. The model was trained to recognize and segment α-syn within images according to a predefined set of models. The results were then integrated into the ZEN image analysis workflow and data extracted. Immunofluorescence labeling was performed by incubating the slides with isothiocyanate or cyanine 3 (Jackson ImmunoResearch Laboratories) for 1 hour. Nuclei were counterstained with DAPI (6-diamidino-2-phenylindole, Invitrogen).

[0116]

[0117] Western blotting

[0118] Proteins were separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). After electrophoresis, the proteins were transferred to polyvinylidene fluoride membranes (Millipore) and blocked with 5% skim milk in phosphate-buffered saline (PBS) containing 0.05% Tween 20 (PBST) for 1 h. The membranes were incubated with primary antibodies overnight at 4°C. After washing with PBST, the membranes were probed with horseradish peroxidase-conjugated secondary antibodies (GenDepot) for 1 h at room temperature. After washing the membranes, immunoblot bands were visualized using an ECL chemiluminescence detection system (GenDepot). The following antibodies were used: anti-α-synuclein (ab212184, Abcam), anti-α-synuclein (610787, BD Biosciences), anti-phosphor-α-synuclein (ab51253, Abcam), anti-TH (T2928, SigmaAldrich), anti-TLR2 (#MA5-32787, Invitrogen), anti-TLR4. (#MA5-16216, Invitrogen), anti-Iba-1 (ab178847, abcam), anti-CD3 (ab16669, abcam), anti-IL-1β (sc-52012, Santa cruz ), anti-iNOS (ab15323, abcam), anti-COX2 (#4842, Cell Signaling), anti-occludin (#91131, Cell signaling), anti-claudin 1 (#37-4900, Invitrogen), anti-claudin 5 (#35-2500, Invitrogen), anti-ChAT (MA5-31383, Invitrogen) and anti-β-actin (#3700, Cell signaling).

[0119]

[0120] Microbiological analysis

[0121] Stool samples were collected 3 months after rotenone treatment and stored in tubes at -20°C before DNA extraction. 16S ribosomal RNA gene sequencing was performed at Macrogen (Korea). Sequencing libraries were prepared according to the Illumina 16S Metagenomic Sequencing Library protocol to amplify the V3 and V4 regions. Input gDNA (5 ng) was PCR amplified using 5x reaction buffer, 1 mM dNTP mix, 500 nM each of the universal F / R PCR primers, and Herculase II fusion DNA polymerase (Agilent Technologies). The first PCR cycle conditions were heat initiation at 95°C for 3 min, followed by 25 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 30 s, and a final extension at 72°C for 5 min. The following universal primer pairs with Illumina adapter overhang sequences were used for the first amplification: 16S Amplicon PCR Forward Primer 5' TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG 3' (SEQ ID NO: 1) and 16S Amplicon PCR Reverse Primer 5' GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC 3' (SEQ ID NO: 2). The first PCR product was purified using AMPure beads (Agencourt Bioscience). After purification, 10 μL of the first PCR product was PCR amplified using NexteraXT Indexed Primer for construction of the final indexed library. The cycling conditions for the second PCR were identical to those for the first PCR except for 10 cycles. The PCR products were purified using AMPure beads.The final purified samples were quantified using qPCR according to the qPCR Quantification Protocol Guide (KAPA Library Quantification Kit for Illumina Sequencing Platforms) and verified using TapeStation D1000 ScreenTape (Agilent Technologies). Sequencing was then performed using the Illumina MiSeq™ platform.

[0122]

[0123] Statistical analysis

[0124] All experiments were independently repeated at least three times, and the results are presented as the mean ± standard deviation (SD) as indicated. Statistical analyses were performed using R, and statistical significance between groups was calculated using the Kruskal-Wallis test and the Conover test with Holm's adjustment. Differences were considered statistically significant at P < 0.05, and 0.05 ≤ P < 0.1 was considered a trend toward significance, which increases sensitivity.

[0125]

[0126] <Result>

[0127] [Example 1] Clinical data

[0128] 1-1. Demographic and clinical characteristics

[0129] PD-DM registered in this example - , PD-DM + / DPP-4i - , PD-DM + / DPP-4i + The demographic and clinical characteristics of the group are shown in Table 1 below.

[0130] [Table 1]

[0131]

[0132]

[0133] Age of symptom onset: PD-DM - PD-DM compared to group + / DPP-4i - and PD-DM + / DPP-4i + was significantly higher in the PD-DM group. There were no differences in disease duration, UPDRS III score, education, CCSIT, or BMI between the three groups. - The group is PD-DM + / DPP-4i - Compared to the PD-DM group, the K-MMSE scores tended to be higher. + / DPP-4i + The group is PD-DM + / DPP-4i - Compared to the group, BDI scores tended to be lower. Among vascular factors, PD-DM - PD-DM over group + The prevalence of hypertension and dyslipidemia was high in the PD-DM group. Vascular risk factors, including hypertension, heart disease, and ischemic stroke, were significantly higher in the PD-DM group. + / DPP-4i - Group and PD-DM + / DPP-4i + Dyslipidemia was similar between groups. PD-DM + / DPP-4i - PD-DM over group + / DPP-4i + It occurred more frequently in the group. There was no significant difference between the groups in other co-prescribed antidiabetic medications, such as metformin, sulfonylurea, thiazolidinedione, alpha-glucosidase, and insulin. The mean WMH score was PD-DM. - It was significantly higher in the PD group with DM compared to the group (9.52±7.62), and PD-DM + / DPP-4i - (13.0±9.17) and PD-DM + / DPP-4i +(12.6±8.11) groups were similar. In terms of motor phenotype, the dominant phenotype did not differ between groups. In addition, all PD-DM + / DPP-4i + Patients were exposed to DPP-4i throughout the entire follow-up period, except for one patient who had been taking DPP-4i for 28 months at the 39-month follow-up point.

[0134]

[0135] 1-2. Comparison of Group Interstriatal DAT Availability

[0136] DAT availability for the entire striatum and each striatal subregion in each group is shown in Table 1 and Figure 1. Regarding subregional striatal DAT availability, PD-DM + / DPP-4i + Group PD-DM + / DPP-4i - Compared with the control group, the PD-DM+ / DPP-4i+ group showed more preserved DAT availability in the anterior putamen (2.56±0.74 vs. 2.10±0.50; P = 0.016), posterior putamen (1.83±0.69 vs. 1.40±0.50; P <0.001), and ventral putamen (1.72±0.58 vs. 1.35±0.37; P = 0.001) (Fig. 3). In other words, Parkinson's disease patients taking DPP-4 inhibitors were confirmed to have less dopaminergic neuron damage compared not only to Parkinson's disease patients not taking DPP-4 inhibitors but also to Parkinson's disease patients without diabetes.

[0137] Also, PD-DM + / DPP-4i + The group is PD-DM -Compared with the PD-DM group, PD-DM showed higher DAT availability in the posterior putamen (1.83±0.69 vs. 1.43±0.59; P<0.001). Multivariate linear regression analysis showed that PD-DM was significantly associated with age at symptom onset, sex, disease duration, hypertension, dyslipidemia, BMI, and total WMH after adjustment for age at symptom onset, sex, disease duration, hypertension, dyslipidemia, BMI, and total WMH. - or PD-DM + / DPP-4i - PD-DM compared to group + / DPP-4i + The group was found to be significantly and independently associated with less severely reduced DAT availability in the whole striatum (β = -0.148, P = 0.011; β = -0.185, P = 0.001), anterior putamen (β = -0.186, P = 0.012; β = -0.207, P = 0.003), posterior putamen (β = -0.336, P <0.001; β = -0.286, P <0.001), and ventral putamen (β = -0.204, P = 0.005; β = -0.250, P <0.001) (Table 2). The absolute values ​​of the standardized coefficients (β) were highest in the posterior putamen model.

[0138] [Table 2]

[0139]

[0140]

[0141] 1-3. Longitudinal evaluation of LED changes between groups

[0142] Six hundred and five PD patients receiving dopaminergic medication for at least 2 years had similar demographic characteristics and striatal DAT availability (Table 3).

[0143] [Table 3]

[0144]

[0145]

[0146] After adjusting for age at symptom onset, sex, hypertension, dyslipidemia, BMI, total WMH, baseline DAT availability in the posterior horn, time, and group × time, there was a significant group × time interaction in the mixed model (p = 0.002), indicating that the longitudinal change pattern of LED differed between groups. PD-DM during the follow-up period + / DPP-4i + The group is PD-DM + / DPP-4i - It was found that lower doses of dopaminergic drugs were required compared to the PD-DM group. + / DPP-4i + The group's LED change is PD-DM - It was confirmed that the increase in dopaminergic drugs was less compared to the group without diabetes (Table 4 and Figure 4). That is, it was confirmed that Parkinson's disease patients taking DPP-4 inhibitors had a smaller increase in dopaminergic drugs compared to not only Parkinson's disease patients not taking DPP-4 inhibitors but also Parkinson's disease patients without diabetes.

[0147] [Table 4]

[0148]

[0149]

[0150] Information on anti-Parkinsonian drugs between groups is shown in Table 5 below.

[0151] [Table 5]

[0152]

[0153]

[0154] 1-4. Development of LID and fatigue by group according to use of DPP-4i

[0155] PD-DM during the follow-up period - Among 484 patients in the group (follow-up period 6.00±2.10 years, number of visits 22.61±9.43), 123 (25.4%) had PD-DM. + / DPP-4i -Among 76 patients (23.7%) in the group (follow-up period 5.45±2.15 years, number of visits 20.21±9.87), and PD-DM + / DPP-4i + Among 46 patients in the PD-DM group (follow-up period, 4.77±1.37 years, number of visits, 16.83±6.07), 2 (4.4%) developed LID. - 135 people (27.9%) in the group, PD-DM + / DPP-4i - 21 people (27.6%) in the group, PD-DM + / DPP-4i + Wearing-off occurred in 3 patients (6.5%) in the group. Kaplan-Meier analysis revealed PD-DM. + / DPP-4i + The group is PD-DM + / DPP-4i - Group and PD-DM - The risk of developing LID was found to be lower compared to the PD-DM group (Plog-rank = 0.047 and 0.039, respectively, Fig. 5a). In terms of fatigue, the Kaplan-Meier analysis showed that PD-DM + / DPP-4i + The group is PD-DM + / DPP-4i - Group and PD-DM - The risk of developing LID was found to be lower in patients with PD-DM compared to the control group (Plog-rank = 0.041 and 0.036, respectively; Fig. 5b). That is, patients with PD taking DPP-4 inhibitors were found to have fewer motor complications (dyskinesia, drug withdrawal) compared not only to patients with PD-DM not taking DPP-4 inhibitors but also to patients with PD-DM without diabetes. After adjusting for covariates, PD-DM + / DPP-4i + The group (HR, 0.294; 95% CI, 0.071–1.213; P = 0.091) had a PD-DM -The risk of developing LID tended to be lower in the PD group (HR, 0.323; 95% CI, 0.099–1.051; P = 0.060) than in the PD group (Table 5). Analysis of patients with diabetes mellitus with PD revealed that PD-DM + / DPP-4i + The group is PD-DM + / DPP-4i - The risk of developing LID was found to be lower in the PD-DM group compared to the control group (HR, 0.194; 95% CI, 0.041–0.907; P = 0.037) (Table 6). However, + / DPP-4i - PD-DM compared to group + / DPP-4i + The HR for the occurrence of fatigue was not significant in the group. The predictability of LID or fatigue was reliable (Harrel's C index ≥ 0.7).

[0156] [Table 6]

[0157]

[0158]

[0159] Finally, it was confirmed that in patients with Parkinson's disease and diabetes, the group taking DPP-4 inhibitors had less dopaminergic neuron loss at the time of diagnosis than the group not taking the drug, and the dopaminergic neuron loss was even milder than the group without diabetes. In addition, when a longitudinal analysis was performed, it was confirmed that the rate of increase in dopaminergic drugs to control Parkinson's symptoms and the rate of occurrence of motor complications were significantly lower in the group taking DPP-4 inhibitors compared to the other groups.

[0160]

[0161] [Example 2] Experimental data

[0162] 2-1. Effects of sitagliptin on the intestines

[0163] The body weights of mice were measured at weeks 1, 3, 5, 6, and 11. There was no significant difference in body weight among the three groups at each time point (Fig. 6A). Intestinal transit, the distance traveled by Evans Blue dye in the intestine, showed a significant difference among the three groups (Kruskal-Wallis test, P = 0.010), and the Rot group showed delayed intestinal transit (P = 0.002) compared to the Ctrl group. The Rot+Sita group showed a relative improvement in intestinal transit delay compared to the Rot group, but the difference was not statistically significant (P = 0.120). Colon length was similar among the three groups (Kruskal-Wallis test, P = 0.978, Fig. 6B). Inflammatory markers Iba-1, TLR2, CD3, and IL-1β were significantly increased in intensity in rotenone-treated mice (Rot) compared to vehicle-treated mice (Iba-1, P <0.001; TLR2, P = 0.032; CD3, P <0.001; IL-1β, P = 0.050). TLR4 levels did not show a significant difference (P = 0.790). The levels of Iba, TLR2, and CD3 in the Rot+Sita group were relatively lower than those in the Rot group (Iba-1, P <0.001; TLR2, P <0.001; CD3, P = 0.001). IL-1β levels were similar but did not reach statistical significance (P = 0.127, Fig. 6c). Immunohistochemical evaluation results showed that rotenone-treated mice had increased numbers of intestinal Iba+, TLR2+, and CD3+ cells compared to vehicle-treated mice. IL-1β was increased in rotenone-treated mice compared to vehicle-treated mice. When sitagliptin was administered together with rotenone (Rot+Sita group), intestinal Iba+, TLR2+, and CD3+ cells and IL-1β were decreased compared to the Rot group (Fig. 6d).Meanwhile, serum inflammatory cytokines (TNF-α, IFN-γ, IL-1β, and IL-6) showed no significant differences among the three groups (Fig. 6e). In terms of intestinal tight junction markers (i.e., Claudin1, Claudin5, and Occludin), Claudin1 and Claudin5 did not differ among the three groups, whereas Occludin was significantly increased in the Rot group compared to the other groups (Fig. 6f).

[0164] Based on the above results, we investigated whether these changes in inflammatory markers were accompanied by α-syn deposition. Immunohistochemistry evaluated intestinal α-syn levels, revealing a higher number of α-syn-positive structures in the mucosa and muscularis propria of rotenone-treated mice (Rot) compared to vehicle-treated mice. Furthermore, coadministration of sitagliptin with rotenone (Rot+Sita group) resulted in improvement (Fig. 7a). Quantitative analysis using 3,3-diaminobenzidine (DAB) intensity showed that α-syn expression in the intestinal mucosa and muscularis propria was increased in rotenone-treated mice compared to vehicle-treated mice (mucosa, P = 0.023; muscle, P = 0.019), and when sitagliptin was administered together with rotenone (Rot+Sita group), it was improved in both the mucosa (P = 0.088) and muscularis propria (P = 0.070) (Fig. 7b). Immunofluorescence analysis using α-syn and cell markers of enteroendocrine cells (EECs) (PYY, GLP-1, Ngn1, and TH) showed that α-syn was expressed in EECs of mouse colonic crypts (Fig. 7c).

[0165]

[0166] 2-2. Cell experiment on the effect of DPP-4 inhibition on α-syn levels and TLR2-mediated inflammatory response

[0167] We analyzed whether rotenone treatment increases α-syn expression in STC-1 cells, an EEC cell line. Rotenone significantly increased α-syn expression in STC-1 cells, which was accompanied by a dose-dependent increase in cleaved PARP. This suggests a dose-dependent increase in apoptosis (Fig. 8a). However, Western blot and immunohistochemical analyses revealed that α-syn protein was not expressed in Caco-2 cells, which resemble intestinal absorptive epithelial cells (Figs. 8b, 8c), consistent with the results of previous animal experiments. Therefore, STC-1 cells were used to analyze the effect of sitagliptin on regulating α-syn levels.

[0168] ELISA analysis in untreated STC-1 cells showed that endogenous α-syn was normally expressed and secreted into the culture medium, and the extracellular and intracellular α-syn levels were 240±58 pg / mL and 9,307±458 pg / mL, respectively, after 48 h of culture. When STC-1 cells were treated with 100 nM rotenone for 48 h, the extracellular α-syn concentration significantly increased to 528±92 pg / mL (P=0.001) and the intracellular concentration to 65,268±1,643 pg / mL (P=0.001). However, in the sitagliptin combination treatment group, the extracellular α-syn concentration was significantly reduced to 439±61 pg / mL (P=0.045), and the intracellular α-syn concentration was significantly reduced to 27,865±6,560 pg / mL (P=0.013) (Fig. 8d).

[0169] Next, we performed cell experiments to determine whether sitagliptin has an immunomodulatory effect on TLR2-mediated gastrointestinal inflammation and changes in α-syn levels. When Raw264.7 cells were co-treated with Pam3CSK4 and sitagliptin, the expression of iNOS (F = 28.91, P < 0.001) and COX2 (F = 14.95, P = 0.005) induced by Pam3CSK4 was significantly inhibited (Fig. 8e). When 100 μM rotenone was treated in enterocyte glial cells (EGCs) for 24 h, the expression of COX2 was significantly increased compared to the control group (P = 0.010), but was significantly decreased when co-treated with sitagliptin (P = 0.012, Fig. 8f). In STC-1 cells, rotenone treatment also increased COX2 expression (P=0.028), but COX2 expression was significantly reduced in the sitagliptin-treated group (P=0.018, Fig. 8g).

[0170]

[0171] 2-3. Effects of sitagliptin on α-syn deposition in the vagus nerve, medulla oblongata, and midbrain

[0172] In a rotenone-induced gut-brain axis-related Parkinson's disease model, we analyzed the effects of sitagliptin on pathological α-syn accumulation in the vagus nerve (VN), dorsal motor nucleus of vagus (DMV), and substantia nigra pars compacta (SNpc).

[0173] Immunohistochemical analysis results showed that the Rot group showed a significant increase in phospho-α-synuclein (p-α-syn) deposition compared to the Ctrl group, and co-administration of sitagliptin in the rotenone-treated group significantly reduced p-α-syn deposition in the VN. Quantitative analysis results also showed that the intensity of p-α-syn immunoreactivity in the VN was significantly increased in the rotenone-treated group compared to the control group (P<0.001), and significantly decreased in the sitagliptin-co-administered group (Rot+Sita) compared to the rotenone-only group (Rot) (P<0.001; Fig. 9a). Immunofluorescence staining using neuronal markers (NF, S100β) confirmed the presence of p-α-syn immunoreactive inclusions in VN neuronal cells (Fig. 9b).

[0174] Similarly, in the DMV, the rotenone-treated group showed increased p-α-syn deposition compared to the control group, and co-administration of sitagliptin inhibited α-syn deposition. Quantitative analysis also showed that the p-α-syn intensity in the DMV of the rotenone-treated group was significantly increased compared to the control group (P<0.001), whereas the p-α-syn intensity in the Rot+Sita group was significantly decreased compared to the Rot group (P=0.003; Fig. 9c). In addition, co-localization of p-α-syn with choline acetyltransferase (ChAT), a cholinergic neuron marker, was confirmed, suggesting that p-α-syn deposition in the DMV is limited to cholinergic neurons (Fig. 9d).

[0175] In the SNpc of the midbrain, p-α-syn intensity was significantly increased in the Rot group compared to the Ctrl group (P<0.001), but was significantly reduced in the Rot+Sita group compared to the Rot group (P=0.004; Figure 5E). In addition, p-α-syn was strongly immunostained in dopaminergic neurons in the SNc of the Rot group, but the immunoreactivity of p-α-syn coexpressed with dopaminergic neurons was confirmed to be reduced in the Rot+Sita group (Figure 9F).

[0176]

[0177] 2-4. Effects of sitagliptin on neuronal loss and motor function in the vagus nerve, medulla oblongata, and midbrain

[0178] We further investigated neuronal loss in the DMV and SNpc. ChAT expression in the DMV was measured by Western blot and was significantly lower in the Rot group compared to the Ctrl group (P=0.034), whereas ChAT expression tended to increase again in the sitagliptin-treated group (P=0.050; Fig. 10a).

[0179] In the SNc of the midbrain, both TH expression and the number of TH-positive (TH+) cells measured by Western blot analysis were significantly reduced in the Rot group compared to the Ctrl group (P=0.010 and P=0.003, respectively). However, although the degree of TH expression reduction between the Rot group and the Rot+Sita group did not differ in the Western blot results (P=0.761), the number of TH+ cells was relatively well preserved in the Rot+Sita group compared to the Rot group (P=0.030; Figs. 10b, 10c).

[0180] Additionally, the average size of microglia was significantly larger in the Rot group than in the Ctrl group (P=0.006), and this change was alleviated after sitagliptin treatment (P=0.032). However, the number of Iba-1+ cells did not differ significantly between the groups (Fig. 10d).

[0181] Finally, the rotarod test results showed that sitagliptin treatment partially restored motor function deficits in rotenone-treated animals (Fig. 10e).

[0182]

[0183] 2-5. Effects of sitagliptin on gut microbiota

[0184] Gut microbiota dysbiosis is known to be closely associated with the development of PD. Fecal pellets were collected at week 12 for microbiota analysis. First, analyses were performed based on alpha and beta diversity. Alpha diversity analysis showed that ASV and Shannon indices were comparable between groups (Fig. 11a). Beta diversity was measured using PcoA, which is based on unweighted UniFrac distances. PERMANOVA analysis showed that the gut microbiota significantly separated among the three groups (F = 2.84, P = 0.001). Similar results were obtained using PcoA, which is based on weighted UniFrac distances (F = 7.86, P = 0.001) (Fig. 11b). The Firmicutes-to-bacteroidetes (F / B) ratio was higher in the Rot+sita group than in the Rot group (P = 0.038) (Fig. 11c). To further investigate how the microbiota structure changed between groups, we analyzed the relative abundance at the genus level. The distribution of fecal microorganisms for all samples at the genus level is shown in Figure 11d, and the relative abundance of microbial taxa at the genus level within the groups is shown in Table 7 below. Boxplots were presented for five species that showed significant differences among the genus-level species (Figure 11e). Compared to the Ctrl group, the genera Prevotella and Alistipes showed relatively high abundances in the Rot group, whereas the genera Clostridium and Ruminiclostridium showed significantly lower abundances. On the other hand, co-treatment with sitagliptin decreased the abundances of Prevotella, Alistipes, and Mediterraneibacter in the Rot + sitagliptin group compared to the Rot group.

[0185] [Table 7]

[0186]

[0187]

[0188] In conclusion, in an animal model of Parkinson's disease, sitagliptin administration reduced intestinal inflammation and decreased α-synuclein, a pathogenic factor in Parkinson's disease, in the intestine, vagus nerve, medulla oblongata, and midbrain. Both animal and cell experiments confirmed that sitagliptin administration downregulates TLR2-associated inflammatory responses in the intestine, thereby reducing α-synuclein expression. Furthermore, sitagliptin administration suppressed neuronal loss in the medulla oblongata and midbrain, partially restored motor ability, and affected the gut microbiome. In summary, sitagliptin improves Parkinson's disease symptoms by blocking the progression of α-synuclein pathology, a major pathogenic factor in Parkinson's disease, from the intestine to the brain, and this effect was demonstrated to be mediated by modulating TLR2-associated inflammatory responses.

[0189]

[0190] While specific aspects of the present invention have been described in detail above, it should be apparent to those skilled in the art that these specific descriptions merely represent preferred embodiments and are not intended to limit the scope of the present invention. In other words, the substantial scope of the present invention is defined by the appended claims and their equivalents.

[0191] Numerical ranges are inclusive of the values ​​defined in the ranges above. Any maximum numerical limit given throughout this specification includes any lower numerical limit, as if that lower numerical limit were explicitly stated. Any minimum numerical limit given throughout this specification includes any higher numerical limit, as if that higher numerical limit were explicitly stated. Any numerical limit given throughout this specification will include any better numerical range within that broader numerical range, as if that narrower numerical limit were explicitly stated.

Claims

1. A pharmaceutical composition for preventing or treating Parkinson's disease, comprising sitagliptin or a pharmaceutically acceptable salt thereof as an active ingredient.

2. A pharmaceutical composition according to claim 1, characterized in that the Parkinson's disease is Parkinson's disease accompanied by type 2 diabetes.

3. A pharmaceutical composition according to claim 1, characterized in that the pharmaceutical composition is for oral administration.

4. A pharmaceutical composition according to claim 1, wherein the sitagliptin suppresses an intestinal inflammatory response.

5. A pharmaceutical composition according to claim 1, wherein the sitagliptin inhibits α-synuclein deposition in the intestine, vagus nerve, medulla oblongata, or midbrain.

6. A pharmaceutical composition according to claim 1, wherein the sitagliptin inhibits neuronal loss in the medulla oblongata and midbrain and restores motor ability.

7. A pharmaceutical composition according to claim 1, characterized in that the pharmaceutical composition has an effect of improving the intestinal microflora of a patient with Parkinson's disease.

8. In the 7th paragraph, the pharmaceutical composition characterized in that the effect of improving the intestinal microflora is a decrease in the abundance of intestinal microorganisms belonging to the genera Prevotella, Alistipes, and Mediterraneibacter.

9. A composition for inhibiting α-synuclein deposition in the intestine, vagus nerve, medulla oblongata or midbrain of an animal model of Parkinson's disease other than a human, comprising sitagliptin as an active ingredient.

10. A method for inhibiting α-synuclein deposition in the intestine, vagus nerve, medulla oblongata, or midbrain, comprising administering sitagliptin to an animal model of Parkinson's disease other than a human.

Images